CN1778953A - Determination of inorganic phosphorus and its diagnostic reagent kit - Google Patents
Determination of inorganic phosphorus and its diagnostic reagent kit Download PDFInfo
- Publication number
- CN1778953A CN1778953A CN 200410065573 CN200410065573A CN1778953A CN 1778953 A CN1778953 A CN 1778953A CN 200410065573 CN200410065573 CN 200410065573 CN 200410065573 A CN200410065573 A CN 200410065573A CN 1778953 A CN1778953 A CN 1778953A
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- Prior art keywords
- reagent
- inorganic phosphorus
- thio
- damping fluid
- diagnosis kit
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- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 86
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 title claims description 49
- 239000011574 phosphorus Substances 0.000 title claims description 49
- 229910052698 phosphorus Inorganic materials 0.000 title claims description 49
- 238000000034 method Methods 0.000 claims abstract description 51
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention is about the measuring method of Inorganic Phosphates and its diagnosis reagent box. Producing hypoxanthine by reacting nucleoside phosphorylase with carnine under the activation ofInorganic Phosphates in the sample of plasma or serum and so on, and producing hydroperoxide by reacting hypoxanthine and xanthine oxidase, and producing acetaldehyde by reacting hydroperoxide with alcohol under the existence of hydroperoxide enzyme, and then transferring oxidized coenzyme to reduced coenzyme by reacting acetaldehyde and aldehyde dehydrogenase. Testing the ascending range of dominant wave-length340nm absorbance and finally measuring the content of Inorganic Phosphates. This method has high specificity and would not be contaminated by material of internal and exogenous sources, and the result is precise and accurate. Diving the diagnosis reagent box into double-dose or three-dose can reduces the cross interaction of each element, keeps the stability of the reagent and deposits chronically. Using this method can realize the fast testing in common ultraviolet/ visible light analyzer or semiautomatic/automatic analyzer and doesn't require special or additional apparatus, so the cost is low. Thus, this method can be easily promoted and applied in the whole industry.
Description
Technical field
The present invention relates to measure the method for inorganic phosphorus, and use the formulated diagnostic kit of this method, belong to medical test determination techniques field.
Background technology
The phosphoric of inside of human body has 87% to be present in the bone approximately, and phosphorus in the blood and the phosphorus in the bone keep dynamic equilibrium relation.On the other hand, certain relation is arranged between the concentration of calcium and phosphorus in the blood, both products keep within limits, are unit with the milligram, and approximately the amassing of the content of calcium and phosphorus is 35～40 in per 100 milliliters of blood plasma.Normal people's blood calcium serium inorganic phosphorus that raises then reduces, and vice versa.When above-mentioned product greater than 40 the time, calcium and phosphorus are deposited on osseous tissue with the bone salts form; Greater than 70 o'clock, metastatic calcification can take place; Less than 35 o'clock, then will hinder the calcification of osseous tissue, even bone salts is dissolved again, influence osteogenesis, cause rickets or richets.Usually, can wait the concentration of regulating calcium and phosphorus in the blood plasma by vitamins D, Rat parathyroid hormone 1-34, thyrocalcitonin.
At present, the phosphorous total amount of human body can't directly be measured, and is that phosphorus acid ion concentration reflects in detection blood plasma or the serum mostly.Because H
2PO
4 -Can stable existence under natural condition such as blood, be the main existence form of inorganic phosphorus, thereby measure H
2PO
4 -Concentration just can represent or know by inference the total amount of inorganic phosphorus indirectly.Detection method commonly used has: phospho-molybdic acid reduction method/non-reduced method, dye binding method, enzyme process, isotopic dilution mass spectrometry, atomic absorption spectrophotometry, flow injection analysis or the like.Wherein, conclusive method is an isotopic dilution mass spectrometry, and the ordinary method that medium laboratory recommends to use is the phospho-molybdic acid colorimetry.
The phospho-molybdic acid reduction method is divided " no proteinemia filtrate method " and " not deproteinate method " again, and the former needs the Deproteinization with blood elder generation, and the latter needs to add nonionic surface active agent to avoid muddy in reagent.The kind of used reductive agent and tensio-active agent is a lot, and the more stable reductive agent of performance has ferrous sulfate, ferrous ammonium sulphate, hydrazonium sulfate, N-methyl p-aminophenol sulfate etc., and tensio-active agent is then the most desirable with polyethylene glycol groups phenyl ether (TritonX-100).
The non-reduced method of phospho-molybdic acid claims " direct ultraviolet method " again, is directly to measure the phospho-molybdic acid complex poly compounds at 340nm or 325nm wavelength, and method is easy, is convenient to automatization.But jaundice, haemolysis, the turbid serum of fat have absorption at 340nm, must do the sample blank, otherwise the result are higher.
Than other detection method, enzyme process is good, highly sensitive with its selectivity, can realize advantage such as automated analysis and enjoy people to favor, and is present people's research focus with the enzymatic assays content of inorganic phosphorus, also is development trend in the future.But, adopting different reagent and test route, the sensitivity of detection, the accuracy of detected result have very big difference, directly have influence on the use and the popularization of this method.
Summary of the invention
The objective of the invention is: a kind of method of enzymatic assays content of inorganic phosphorus is provided, and uses the formulated inorganic phosphorus diagnosis kit of this method.Adopt the reagent in this test kit, can utilize ultraviolet analyser or semi-automatic/automatic clinical chemistry analyzer that the content of inorganic phosphorus in the samples such as blood plasma, serum is carried out fast, accurately measures, so that promote the use of this detection method and diagnostic reagent, make in this area the medical test means of testing obtain abundant and development.
For realizing purpose of the present invention, a kind of method of measuring inorganic phosphorus, undertaken by following steps:
At first,, make it to take place following reaction with needs such as blood plasma, serum sample of measuring and the reagent mixing that contains inosine, Phosphatase, nucleotide, XOD, ethanol, catalase, aldehyde dehydrogenase and oxidized coenzyme,
Then, reaction mixture is placed under ultraviolet analyser or half, the automatic clinical chemistry analyzer, detect the ascensional range of the absorbancy of predominant wavelength 340nm, and then calculate the content of inorganic phosphorus in the sample.
In the mensuration process, the usage ratio of sample and reagent by volume was controlled at 1: 10～1: 500, and temperature of reaction is controlled at 20 ℃～50 ℃, and the reaction times was controlled at 2～30 minutes, set during detection commplementary wave length more than 405nm to play the interferential effect of removing.
Realize that it can be single agent that the inventive method is measured the diagnostic kit of content of inorganic phosphorus in the samples such as blood plasma, serum, is grouped into by following one-tenth:
Damping fluid 40～200mmol/l,
Inosine 0.2～10mmol/l,
Ethanol 1～30mmol/l,
Oxidized coenzyme 0.5～20mmol/l,
Phosphatase, nucleotide 500～50000U/l,
XOD 500～50000U/l,
Catalase 500～50000U/l,
Aldehyde dehydrogenase 500～50000U/l,
Stablizer/reagent cumulative volume 10～80%.
Also the various compositions in above single agent can be made up, be mixed with two agent, such as:
| Composition | Content range | |
| Reagent I | Damping fluid | 40～200mmol/l |
| Ethanol | 1～30mmol/l | |
| Oxidized coenzyme | 0.5～20mmol/l | |
| Phosphatase, nucleotide | 500～50000U/l | |
| XOD | 500～50000U/l | |
| Catalase | 500～50000U/l |
| Aldehyde dehydrogenase | 500～～50000U/l | |
| Stablizer/reagent I cumulative volume | 10～80％ | |
| Reagent II | Damping fluid | 40～200mmol/l |
| Inosine | 0.2～10mmol/l | |
| Stablizer | 10～50mmol/l |
The prescription of two agent is not limited only in the above-mentioned table listed, the wherein composition of reagent I---ethanol, oxidized coenzyme, Phosphatase, nucleotide, XOD, catalase, aldehyde dehydrogenase etc., can be placed on reagent II, inosine among the reagent II also can be put into reagent I, so can form multiple formulations, enumerate no longer one by one.
For making reagent more stable, can also be mixed with three doses, such as:
| Composition | Content range | |
| Reagent I | Damping fluid | 40～200mmol/l |
| Ethanol | 1～30mmol/l | |
| Oxidized coenzyme | 0.5～20mmol/l | |
| Stablizer | 10～50mmol/l | |
| Reagent II | Damping fluid | 40～200mmol/l |
| Phosphatase, nucleotide | 500～50000U/l | |
| XOD | 500～50000U/l | |
| Catalase | 500～50000U/l | |
| Aldehyde dehydrogenase | 500～50000U/l | |
| Stablizer/reagent II cumulative volume | 10～80％ | |
| Reagent III | Damping fluid | 40～200mmol/l |
| Inosine | 0.2～10mmol/l | |
| Stablizer/reagent III cumulative volume | 10～80％ |
Similar with two agent, three doses prescription also is not limited only to above-mentioned prescription, wherein the ethanol of reagent I, oxidized coenzyme can be placed among reagent II or the reagent III, Phosphatase, nucleotide among the reagent II, XOD, catalase, aldehyde dehydrogenase etc. can be placed among reagent I or the reagent III, inosine among the reagent III also can be put among reagent I or the reagent II, so can form multiple formulations, enumerate no longer one by one.
In the middle of the composition of above-mentioned diagnostic kit, selecting the basic demand of damping fluid is that pH value is within 6.0～11.0 scopes, can be: " Tutofusin tris～hydrochloric acid (Tris-HCl) damping fluid ", " trolamine (Triethanolamine) damping fluid ", " 2-amino-2-methyl-1-propanol (2-Amino-2-methyl-1-propanol) damping fluid ", " imidazoles～hydrochloric acid (Imidazole-HCl) damping fluid ", " glycylglycine (Glycylglycine) damping fluid ", " citric acid～sodium citrate buffer solution ", " Veronal sodium～hydrochloride buffer ", " boric acid～borate buffer solution ", " glycine～sodium hydrate buffer solution ", at least a in " borax～sodium hydrate buffer solution " or " yellow soda ash～sodium bicarbonate buffer liquid ", but range of choice be not subjected to these enumerate limit.
In addition, for reducing the cross influence between each reagent composition, the stability of maintenance reagent, so that standing storage, usually add stablizer in the middle of the reagent I/ reagent II of above single agent, two agent, three doses the reagent I/ reagent II/ reagent III, its consumption accounts for 10～80% (perhaps concentration is within 10～50mmol/l scopes) of place reagent cumulative volume.
Material with used as stabilizers can be: at least a in ethylene glycol, propylene glycol, glycerine, ammonium sulfate, thioglycol, adenosine diphosphate (ADP), bovine serum albumin, carbonate, cholate, dextran, ethylenediamine tetraacetic acid (EDTA), flavin adenine dinucleotide, vitamin B2 phosphate, glucose, glutaminate, reduced glutathion, lactose, N.F,USP MANNITOL, succinate or the sodium-chlor.
In the middle of the reagent composition of above-mentioned inorganic phosphorus diagnosis kit, described oxidized coenzyme is---NAD NAD
+, NADP+ NADP
+, thio-NAD+ thio-NAD
+Perhaps thio-NADP+ thio-NADP
+Described reduced coenzyme is---nicotinamide adenine dinucleotide reduced NADH, NADPH NADPH, Thio-NADH thio-NADH or thio-NADPH thio-NADPH.
No matter studies show that, take all factors into consideration from the accuracy of measurement result and economy two aspects of preparation cost, be single agent, two agent or three doses, and the inorganic phosphorus diagnosis kit of following system component relation is comparatively desirable, also is preferred version of the present invention:
Damping fluid 80～120mmol/l,
Inosine 1～5mmol/l,
Ethanol 3～12mmol/l,
Oxidized coenzyme 1～5mmol/l,
Phosphatase, nucleotide 5000～10000U/l,
XOD 5000～10000U/l,
Catalase 5000～10000U/l,
Aldehyde dehydrogenase 5000～10000U/l,
Stablizer/reagent cumulative volume 10～80%.
The present invention utilizes under the activation of the inorganic phosphorus of Phosphatase, nucleotide in samples such as blood plasma, serum and the inosine effect, xanthoglobulin that generates and XOD reaction generate hydrogen peroxide, hydrogen peroxide generates acetaldehyde with ethanol synthesis under catalatic effect, acetaldehyde is reacted into reduced coenzyme with oxidized coenzyme again under the effect of aldehyde dehydrogenase.Because reduced coenzyme has very significantly absorption peak at 340nm, and the oxidized coenzyme of their correspondences does not have absorption peak at 340nm, therefore, measures the ascensional range of reaction front and back 340nm absorbancy, just can reflect the content of inorganic phosphorus in the sample well.
The outstanding substantive distinguishing features and the obvious improvement of technical solution of the present invention mainly shows:
(1) the present invention utilizes Enzymology method fully, enzyme digestion reaction has the high characteristics of specificity, by oxidized coenzyme is reacted into reduced coenzyme, quantitative response goes out the content of inorganic phosphorus in the sample, and test result is (stability of oxidized coenzyme in solution is high more a lot of than reducibility coenzyme) accurately;
(2) reacted constituent of participation enzyme linked reaction system all adds, and is not subjected to the pollution of inside and outside source material, test process tolerance range height;
(3) this method is easy, easy to operate, can obtain detected result fast, and the reaction be under buffer conditions, to carry out, do not pollute the environment;
(4) this method just can rapid detection on general ultraviolet/visible light analysis instrument or half, automatic clinical chemistry analyzer, does not need special or additional instruments, and testing cost is cheap, is convenient to apply in the industry;
(5) use the reagent that measuring method provided by the invention can be made various ways such as liquid reagent, powdered reagent, dried reagent, be mainly used in the content of measuring serium inorganic phosphorus in human body and other animal body, also can be, the content that supplementary means is measured inorganic phosphorus in other sample such as concentrate in conjunction with dilution;
(6) liquid inorganic phosphorus diagnosis kit provided by the invention, good stability, the three-D space structure that is present in wherein each kind of enzyme is kept perfectly, and has guaranteed the application testing effect well.Make after two agent or three doses, can further reduce the cross influence between the various compositions, improve the stability of reagent, be convenient to standing storage.
Embodiment
Below in conjunction with specific embodiment technical solution of the present invention is described further.These examples only are some exemplary applications, can not be interpreted as a kind of restriction to claim protection domain of the present invention.
Embodiment one (single agent)
Prepare inorganic phosphorus diagnosis kit in the serum by following composition and consumption:
Glycylglycine damping fluid 80mmol/l,
Inosine 1mmol/l,
Ethanol 3mmol/l,
thio-NAD
+ 1mmol/l，
Phosphatase, nucleotide 5000U/l,
XOD 5000U/l,
Catalase 5000U/l,
Aldehyde dehydrogenase 5000U/l,
Ethylene glycol 50% (accounting for the reagent cumulative volume).
Go up to set at automatic clinical chemistry analyzer (Hitachi-7080): 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, the volume ratio of sample and reagent is 1: 25, the Direction of Reaction is positive reaction (absorbancy rises, down together).
Adding serum sample and the single agent that is made into, both detect, write down the rising situation of 340nm absorbancy at the inner mixing automatically of analyser.Detect altogether and read a little 31 times, approximately every 20 seconds record one secondary data, according to methods such as rate method, end-point methods, the contrast corresponding standard curve calculates the content of inorganic phosphorus in the serum sample.Same batch sample is repeatedly repeated above process, test result good reproducibility, tolerance range height.
Embodiment two (two agent)
Prepare inorganic phosphorus diagnosis kit in the serum by following composition and consumption:
Reagent I---
Imidazoles～hydrochloride buffer 100mmol/l,
Ethanol 8mmol/l,
NADP
+ 3mmol/l，
Phosphatase, nucleotide 8000U/l,
XOD 8000U/l,
Catalase 8000U/l,
Aldehyde dehydrogenase 8000U/l,
Glycerine 50% (accounting for reagent I cumulative volume);
Reagent II---
Imidazoles～hydrochloride buffer 100mmol/l,
Inosine 3mmol/l,
Ethylene glycol 20mmol/l.
Go up to set at automatic clinical chemistry analyzer (Hitachi-7080): 30 ℃ of temperature, 15 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, sample is 1: 25 with the ratio of the cumulative volume of reagent I and reagent II, reagent I is 4: 1 with the ratio of the consumption of reagent II, and the Direction of Reaction is positive reaction.
Adding serum sample and reagent I add reagent II after 5 minutes earlier, and the three detects, writes down the rising situation of 340nm absorbancy at the inner mixing automatically of analyser.According to methods such as rate method, end-point methods, the contrast corresponding standard curve calculates the content of inorganic phosphorus in the serum sample.Same batch sample is repeatedly repeated above process, test result good reproducibility, tolerance range height.
Embodiment three (three doses)
Prepare inorganic phosphorus diagnosis kit in the serum by following composition and consumption:
Reagent I---
Trolamine damping fluid 120mmol/l,
Ethanol 12mmol/l,
thio-NADP
+ 5mmol/l，
Propylene glycol 20mmol/l;
Reagent II---
Trolamine damping fluid 120mmol/l,
Phosphatase, nucleotide 10000U/l,
XOD 10000U/l,
Catalase 10000U/l,
Aldehyde dehydrogenase 10000U/l,
Propylene glycol 50% (accounting for reagent II cumulative volume);
Reagent III---
Trolamine damping fluid 120mmol/l,
Inosine 5mmol/l,
Ethylene glycol 20mmol/l.
Go up to set at automatic clinical chemistry analyzer (Hitachi-7080): 25 ℃ of temperature, 20 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, sample is 1: 25 with the ratio of the cumulative volume of reagent I, reagent II and reagent III, the consumption of reagent I, reagent II and reagent III is 8: 1: 1, and the Direction of Reaction is positive reaction.
Adding serum sample and reagent I, reagent II add reagent III after 5 minutes earlier, and they detect, write down the rising situation of 340nm absorbancy at the inner mixing automatically of analyser.According to methods such as rate method, end-point methods, the contrast corresponding standard curve calculates the content of inorganic phosphorus in the serum sample.Same batch sample is repeatedly repeated above process, test result good reproducibility, tolerance range height.
Embodiment four (two agent preference)
Prepare the serium inorganic phosphorus diagnostic kit by following composition and consumption:
Reagent I---
Tris-HCl damping fluid 100mmol/l,
Inosine 1mmol/l,
Ethanol 3mmol/l,
NAD
+ 2mmol/l，
Glycerine 20mmol/l;
Reagent II---
Tris-HCl damping fluid 100mmol/l,
Phosphatase, nucleotide 6000U/l,
XOD 7000U/l,
Catalase 8000U/l,
Aldehyde dehydrogenase 9000U/l,
Glycerine 50% (accounting for reagent II cumulative volume).
Go up to set at automatic clinical chemistry analyzer (Hitachi-7080): 37 ℃ of temperature, 10 minutes reaction times, test predominant wavelength 340nm, more than the test commplementary wave length 405nm, sample is 1: 25 with the ratio of the cumulative volume of reagent I and reagent II, reagent I is 4: 1 with the ratio of the consumption of reagent II, and the Direction of Reaction is positive reaction.
Add serum sample and reagent I earlier, add reagent II after 5 minutes, following principle reaction takes place at the inner mixing automatically of analyser in the three:
Detect, write down the rising situation of 340nm absorbancy.According to methods such as rate method, end-point methods, the contrast corresponding standard curve calculates the content of inorganic phosphorus in the serum sample.Same batch sample is repeatedly repeated above process, test result good reproducibility, tolerance range height.
In a word, experimental results show that and adopt measuring method of the present invention, can pass through ultraviolet analyser or semi-automatic/automatic clinical chemistry analyzer device fully, determine the content of inorganic phosphorus in the samples such as blood plasma, serum, measurement sensitivity height, tolerance range are good, are not subjected to the pollution of inside and outside source material.And, inorganic phosphorus diagnosis kit provided by the invention, good stability still can accurately detect the content of inorganic phosphorus in all kinds sample after the long storage time.
Claims (9)
1. method of measuring inorganic phosphorus may further comprise the steps:
1. with testing sample and the reagent mixing that contains inosine, Phosphatase, nucleotide, XOD, ethanol, catalase, aldehyde dehydrogenase and oxidized coenzyme, make it to take place following reaction,
2. reaction mixture is placed under ultraviolet analyser or the semi-automatic/automatic clinical chemistry analyzer, detect the ascensional range of the absorbancy of predominant wavelength 340nm, calculate the content of inorganic phosphorus in the sample.
2. the method for mensuration inorganic phosphorus according to claim 1 is characterized in that: the usage ratio of testing sample and reagent by volume was controlled at 1: 10～1: 500.
3. the method for mensuration inorganic phosphorus according to claim 1 and 2 is characterized in that: temperature of reaction is controlled at 20 ℃～50 ℃, and the reaction times was controlled at 2～30 minutes, sets commplementary wave length during detection more than 405nm.
4. inorganic phosphorus diagnosis kit, reagent is grouped into by following one-tenth in the box:
Damping fluid 40～200mmol/l,
Inosine 0.2～10mmol/l,
Ethanol 1～30mmol/l,
Oxidized coenzyme 0.5～20mmol/l,
Phosphatase, nucleotide 500～50000U/l,
XOD 500～50000U/l,
Catalase 500～50000U/l,
Aldehyde dehydrogenase 500～50000U/l,
Stablizer/reagent cumulative volume 10～80%.
5. inorganic phosphorus diagnosis kit according to claim 4 is characterized in that: the PH scope of described damping fluid is 6.0～11.0.
6. inorganic phosphorus diagnosis kit according to claim 5 is characterized in that: described damping fluid is " Tutofusin tris～hydrochloride buffer ", " trolamine damping fluid ", " 2-amino-2-methyl-1-propanol damping fluid ", " imidazoles～hydrochloride buffer ", " glycylglycine damping fluid ", " citric acid～sodium citrate buffer solution ", " Veronal sodium～hydrochloride buffer ", " boric acid～borate buffer solution ", " glycine～sodium hydrate buffer solution ", at least a in " borax～sodium hydrate buffer solution " or " yellow soda ash～sodium bicarbonate buffer liquid ".
7. inorganic phosphorus diagnosis kit according to claim 4 is characterized in that: described stablizer is at least a in ethylene glycol, propylene glycol, glycerine, ammonium sulfate, thioglycol, adenosine diphosphate (ADP), bovine serum albumin, carbonate, cholate, dextran, ethylenediamine tetraacetic acid (EDTA), flavin adenine dinucleotide, vitamin B2 phosphate, glucose, glutaminate, reduced glutathion, lactose, N.F,USP MANNITOL, succinate or the sodium-chlor.
8. inorganic phosphorus diagnosis kit according to claim 4 is characterized in that: described oxidized coenzyme is---NAD NAD
+, NADP+ NADP
+, thio-NAD+ thio-NAD
+Perhaps thio-NADP+ thio-NADP
+Described reduced coenzyme is---nicotinamide adenine dinucleotide reduced NADH, NADPH NADPH, Thio-NADH thio-NADH or thio-NADPH thio-NADPH.
9. any inorganic phosphorus diagnosis kit in the claim 4～8 is characterized in that: described reagent is made into single agent, two agent or three doses.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112481354A (en) * | 2019-09-11 | 2021-03-12 | 上海云泽生物科技有限公司 | Mycophenolic acid detection reagent and preparation method thereof |
| CN114773416A (en) * | 2022-04-01 | 2022-07-22 | 深圳瑞亚力集团有限公司 | NADH protective agent and preparation method thereof |
-
2004
- 2004-11-23 CN CN 200410065573 patent/CN1778953A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112481354A (en) * | 2019-09-11 | 2021-03-12 | 上海云泽生物科技有限公司 | Mycophenolic acid detection reagent and preparation method thereof |
| CN114773416A (en) * | 2022-04-01 | 2022-07-22 | 深圳瑞亚力集团有限公司 | NADH protective agent and preparation method thereof |
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